How to control pH in electrophoresis-----the problem of the buffer - (Jun/09/2006 )
Hi, I am a new comer.
Now I am trying to study the mobility of DNA in different pH by electrophoresis. I hope to show the change of DNA charges in different pH. I hope to check from pH2--pH8.
1. What kind of running buffer can be used? Of course I hope to employ only one type to buffer if possible.
2. Is it necessary to prepare the gel with the same buffer at the desired pH? Some one said that run the eletrophoresis for an hour in the buffer (for example pH4), the pH of the gel will change to pH4, I do not know whether it is true.
3. Is it OK to run electrophoresis while there is much Cl- in the running buffer? I am afraid that Cl2 will be produced to cause danger. Am I right?
Buffers are only effective in holding pH when used near the pKa of the compound used. Look up "Good buffers" (named after Dr. Good, not only because they work well). There is a wide variety with different pKa's. You'll need at least 3-4 different ones to cover that range. If you want to avoid preparing gels with different buffers, you can prepare gels in water and then soak in excess buffer for a few hours. This would be better than making it with one buffer and then trying to switch it. You'll dilute the buffer a bit in doing this, but it probably won't matter much. People avoid chlorine ions in electrophoresis buffers primarily to reduce corrosion of the wiring -- but most of it is platinum anyway. You definitely don't need to worry about poisoning yourself with chlorine, but it would be better to use acetic acid to neutralize basic buffers, and sodium hydroxide to neutralize acidic buffers. You should read one of the online tutorials on buffer composition and use, such as here:
Looking at this chart, you might get away with using Tris, acetic, citric, and phosphoric acid, which would probably by relatively easy for you to obtain.
Check out this buffer capacity discussion - it may be enough if you look at the plot.